Previous studies have shown that STAT4 is necessary for CD4+ Th1 differentiation but has not addressed the question of the role of this factor in Th2 differentiation. In particular, while they showed that STAT4 has a role independent of maintenance of the IL-12R b2, they did not address the question of whether STAT4 is the intra-cellular factor whose regulation determines b2 chain expression and thus the course of T cell differentiation. Recognizing that the answer to this question would require cells that express constitutive b2 chain that thus allow studies of the effect of perturbation of STAT4 expression in the setting of constant b2 chain expression, we realized that we were in a unique position to approach this problem. In initial studies along these lines we showed that BALB/c double transgenic T cells expressing both a transgene for OVA TCR and the b2 chain (as described above) that was stimulated under Th1 conditions and then infected with a GATA-3 expressing retrovirus underwent a shift in T cell differentiation form a Th1 cell producing IFN-g to a Th2 cell producing mainly IL-4. Moreover, such cells manifested greatly decreased STAT4 phosphorylation. Similar results were obtained with cells from single transgenic mice not bearing a b2 chain transgene. These striking results were cell autonomous in that extensive studies showed that they did not depend on the presence of exogenous Th2 cytokines. These studies indicated that, in keeping with the results described above, Th2 differentiation did not depend on down-regulation of the b2 chain and added the new fact that such differentiation resulted in decreased STAT4 activation. In addition, they were corroborated by subsequent studies showing that Th2 cells exhibit strikingly reduced STAT4 levels and that STAT4 levels and GATA-3 levels in develping Th1 and Th2 cells (as determined by real-time PCR) are reciprocal. At this point, we investigated the role of T-bet in the T cell differentiation process since the newly described transcription factor has been shown to play a major role in Th1 differentiation. In these studies, we showed that infection of cells with a STAT4-expressing retrovirus did not induce increased T-bet levels in either Th1 or Th2 cells so that Th2 differentiation induced by GATA-3 (associated with decreased STAT4) could not be ascribed to a failure to maintain T-bet concentration. Nevertheless, T-bet levels were depressed in cells infected with a retrovirus expressing GATA-3 and infection of Th2 cells with a T-bet expressing retrovirus induced Th1 differentiation. Then, in further studies, we studied double transgenic T cells infected with two retroviruses using a unique approach of labeling separate retroviruses with GFP and YFP and then sorting for cells exhibiting both green and yellow fluorescence. Here we showed that Th1 cells infected with both GATA-3 and STAT4 do not undergo a shift to Th2 differentiation, whereas cells infected with both GATA-3 and T-bet do not undergo this shift. These studies show rather clearly that expression of STAT4, but not T-bet, stands in the way of Th2 differentiation induced by GATA-3 and that STAT4 must be down-regulated to achieve Th2 differentiation. In a final series of studies, we infected single and double transgenic developing Th2 cells with a STAT4-expressing retrovirus and showed that stimulation of these cells in the presence of IL-12 shifted them toward Th1 differentiation in that infected cells produced vastly increased amounts of IFN-g and somewhat decreased amounts of IL-4. While, as expected, the effect was more pronounced in cells bearing a b2 transgene, it also occurred in cells without this transgene and in the latter case it was accompanied by the appearance of low levels of endogenous b2 chain. Whether this is a direct effect of STAT4 on b2 chain expression or an indirect effect occurring via T-bet expression remains to be seen. These studies establish for the first time that STAT4 is a central player in the molecular events controlling T cell differentiation in that they show that inducing Th2 differentiation GATA-3 must down-regulate STAT4. Going back to the original question of the relation of STAT4 to the IL-12R b2 chain several possibilities must be considered. One is that STAT4 regulates the b2 chain directly and is thus the true molecular switch regulating T cell differentiation. A second is that STAT4, in that it controls initial IFN-g production, regulates T-bet via STAT1 and T-bet, in turn, regulates the b2 chain; in this case, STAT4 regulates the b2 chain only indirectly. A third possibility is that both scenarios are true and that both STAT4 and T-bet are necessary for b2 chain expression. Further studies with T-bet deficient cells can answer this question.